The discovery of synthetic small molecules and chemical reactions with desirable features are central activities of chemistry. Current approaches to small-molecule synthesis, small-molecule discovery, and reaction discovery have proven to be fruitful but are also limited in fundamental ways. Several years ago our laboratory initiated a program to develop a new approach to the synthesis and discovery of small molecules that combines powerful aspects of natural biosynthesis and molecular evolution with the flexibility of synthetic organic chemistry. We discovered that DNA duplex formation exerts remarkable control over the effective molarity of DNA-linked reactants, including those unrelated in structure to DNA. This surprising generality of DNA-templated organic synthesis has enabled us to subject DNA sequences encoding synthetic molecules to translation, selection, and amplification that parallel the evolution of biological molecules in nature. During the original granting period, we developed the foundations of DNA-templated synthesis, explored several of its unique modes of controlling activity, and developed in vitro selections of DNA-linked small molecules. We integrated these developments to enable the synthesis and selection of a small pilot library of DNA-templated macrocycles and an intermediate library of DNA-templated heterocycles, and confirmed that a heterocycle discovered by library selection has affinity for its protein target. Most recently, we have completed the synthesis and initial characterization of a 13,824-membered DNA-templated macrocycle library. In addition, we exploited unique features of DNA-templated synthesis and in vitro selection to develop a novel approach to the discovery of bond-forming reactions, and used this approach in an early-stage effort to discover a Pd(II)-mediated oxidative alkyne-alkene coupling reaction. We recently extended this work by developing a DNA-encoded reaction discovery system that is compatible with reaction conditions that do not support DNA hybridization (including organic solvents and high temperatures). Using this second-generation system, we discovered a Au(III)- or triflic acid-mediated alkene hydroarylation reaction. In this proposal we describe the large-scale, systematic application of the capabilities that emerged during the first granting period to realize the potential of DNA- and selection-based approaches for small-molecule synthesis, small-molecule discovery, and reaction discovery. We propose the functional selection of our recently synthesized DNA-templated macrocycle libraries for the ability to interact with protein and RNA targets of biological interest, and the development of new DNA- and selection-based methods for the discovery of chemical reactions, including stereoselective transformations. The discoveries that emerge from work proposed here may provide new molecular probes and therapeutic leads for biological pathways relevant to human disease, as well as new chemical reactions that augment the ability of scientists to make molecules of interest. PUBLIC HEALTH RELEVANCE: Our proposed work integrates powerful features of living systems such natural selection and DNA replication with test-tube chemistry to create new ways of discovering manmade molecules and chemical reactions with tailor-made properties. The molecules and reactions discovered in this work, together with the insights gained from their use, may lead to new medicines and research tools.